In bacteria, the 450 kDa RNA polymerase (RNAP) holoenzyme, comprising the evolutionarily conserved catalytic core (subunit composition 2') combined with the initiation-specific subunit, directs transcription initiation. Bacterial transcription depends on a primary factor that is essential for viability, as well as alternative 's that control specific regulons. A major mechanism to control transcription initiation is through regulation of activity. Dramatic insights have come from structural studies of 's and holoenzymes. Nevertheless, many challenges remain. In this competing continuation, we propose studies to further our understanding of factor structure and function, and interactions with accessory factors. Specifically, we propose to: 1. Determine the structural basis for interactions with the -10 element in the initiation of promoter melting. We will determine crystal structures of complexes between and in vitro- selected DNA aptamers that mimic /-10 element interactions critical for promoter melting. 2. Map the position of E. coli 701.1 on the RNAP holoenzyme during different stages of promoter open complex formation. A protein-protein crosslinking/mapping approach (guided by our new structure of 1.1) will be used. The crosslinking-based data will be combined with existing FRET-based distance constraints to generate structural models of 701.1 in the context of the RNAP holoenzyme in holoenzyme alone, as well as in closed and open promoter complexes. 3. Structurally and functionally characterize /anti- complexes: Staphylococcal aureus phage G1 ORF67. S. aureus phage G1 ORF67 binds to S. aureus A domain 4 and is a potent inhibitor of S. aureus transcription. We will determine crystal structures of complexes between phage G1 ORF67 and S. aureus A domain 4, and functionally characterize the mechanism of G1 ORF67 inhibition of S. aureus transcription. 4. Determine the structural basis for N interactions with its promoter DNA. We will determine the crystal structure of a complex between Aquifex aeolicus N and promoter DNA.